J. Am. Chem. SOC.1994,116,9365-9366
9365
Application of Photoisomerizable Antigenic Monolayer Electrodes as Reversible Amperometric Immunosensors Itamar Willner,' Ron Blonder, and Arie Dagan Institute of Chemistry and Farkas Center for Light-induced Processes The Hebrew University of Jerusalem Jerusalem 91904,Israel
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Figure 1. Organization of a dinitrophenyl antigen monolayer on an Au electrode.
Received June 17, 1994 Antibody-antigen interactions play a major role in biosensor technology.lJ The strong antigen-antibody interactions limit the applicability of antigen (or antibody) sensing surfacestosinglecycle analytic devices. Conversionof single-cycleimmunosensors to multicycle devices is expected to provide a scientific and technological breakthrough in biosensor technology. Here we report on the development of amperometric immunosensors by the organization of antigenic self-assembledmonolayer electrodes and on the use of photoisomerizableantigen monolayer electrodes as an approach to develop reversible amperometric immunosensors. Modification of biomaterials by photoisomerizable units3 provided a means to photostimulate "on-off biocatalytic and binding properties of enzymes4 and proteins.5 Photoregulated binding a trans-azobenzene hapten to the respective antibody6a and selective association of a dinitro spiropyran with the antidinitrophenyl (anti-DNP) antibody6b have revealed selective association of photoisomerizableantigens with their antibodies.6 Development of amperometric biosensors has been examined extensively in recent years? and redox modified enzymemonolayer electrodes were applied as amperometric sensors.8.9 The development of the amperometric biosensor is based on the construction of a self-assembled antigen monolayer on the electrode surface. This electrode electrically communicates with a redox probe, R+/R, in the electrolyte solution and provides an amperometric response. Interaction of the electrode with the antibody, Ab, results in binding of the Ab to the monolayer. The electrode surface is then insulated toward the solubilized redox probe, and its amperometric response decreases. The extent of electrode insulation is controlled by the Ab concentration and the time of incubation of the monolayer electrode with the Ab solution. Figure 1 shows the stepwise construction*of the antigen monolayer toward the DNP-Ab. The amperometric response of the antigen monolayer electrode in an electrochemical cell that included K3Fe(CN)6(1) as redox probe was recorded at different DNP-Ab concentrations. The difference between the amperometric signal of the antigen electrode and that of the antigen electrode in the presence of DNP-Ab, Ai,, increased linearly as (1) Scheller, F.; Schubert, F. Techniquesandlnstrumentation in Analytical Chemistry, Vol. 11: Biosensors; Elsevier: Amsterdam, 1992; pp 275-284. (2) Anglister, J.; Jacob, C.; Assulin, 0.;Ast, G.;Pinker, R.; Arnon, R. Biochemistry 1988, 27, 717. (3) Willner, I.; Willner, B. In Bioorganic Photochemistry-Biological Applications of Photochemical Switches; Morrison, H., Ed.; Wiley: New York, 1993; pp 1-110. (4) Willner, I.; Rubin, S.; Riklin, A. J. Am. Chem. SOC.1991,113, 4013. ( 5 ) Willner, I.; Rubin, S.;Wonner, J.; Effenberger, F.; Biuerle, P. J. Am. Chem. Soc. 1992, 114, 3150. ( 6 ) (a) Harada, M.; Sisido, M.; Hirose, J.; Nakanishi, M. FEBS Lett. 1991,286,6. (b) Willner, I.; Blonder, R.; Dagan, A. J.Am. Chem. SOC.1994, 116, 3121. (7) (a) Wilson, G.Biosensors: Fundamentals and Applications; Turner, A,, Karube, I., Wilson, G., Eds.; Oxford Univ. Press: New York 1987. (b) Heller, A. Acc. Chem. Res. 1990, 23, 128. (8) Willner, I.; Katz, E.; Riklin, A,; Kasher, R. J. Am. Chem. SOC.1992, 114, 10965. (9) Willner, I.; Riklin, A,; Shoham, B.; Rivenzon, D.; Katz, E. Ado. Mater. 1993, 5, 912.
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Figure 2. Schematic configuration of a reversible amperomctric immunosensor utilizing a photoisomerizable monolayer electrode.
the DNP-Ab concentration was elevated, and Ab content as low as 0.5 pM could be detected by this monolayer antigen electrode.10 The principle of reversible amperometric immunosensors is shown in Figure 2. The antigen monolayer consists of a photoisomerizable component. In one photoisomer state (state A), the antigen recognizes the Ab, and the difference in the amperometricresponses of the Ab-linked electrodeand theantigen monolayer electrode, lacking the Ab, toward the solubilized redox couple provides a quantitative measure for the Ab concentration. Upon completion of the measuring cycle the antigen monolayer is photoisomerized to state B. This leads to distortion of the antigen monolayer,and the perturbed surface lacks affinity toward the Ab. The Ab is washed off and the monolayer electrode is further illuminated (huz) to restore the active antigen monolayer electrode. Thus, a two-step illumination procedure regenerates the active antigen electrode. Transformation of the DNP-Ab amperometric immunosensor into a reversible multicycle sensing device was accomplished by organization of a photoisomerizable dinitrophenyl spiropyran antigen monolayer electrode, Scheme 1. The surface density of thedinitrospiropyranresiduescorrespondsto5.0 X 10-11mol.cm-2. The resulting 4a dinitro spiropyran monolayer electrode exhibits reversible photoisomerizable properties.12 Illumination of the 4a monolayer electrode, X = 360-380 nm, results in the 4b monolayer electrode, and further irradiation of the 4b monolayer electrode, X > 495 nm, restores the 4a monolayer. The amperometric response of 4a monolayer electrodesin the presence (10) The antigen monolayer electrode also provides a means to analyze the antigen itself. Challengingof the monolayer electrode with an analytesolution consisting of a constant concentration of the DNP-Ab and variable concentrations of antigen 2 results in competitive association of the Ab to the monolayer electrode. At high analyte concentrations of 2 the monolayer electrode is not insulated due to binding of the Ab to the antigen analyte. Using this approach, the monolayer antigen electrode was sensitive enough to detect up to 0.5 X 1 V M of the antigen ( 2 ) . (1 1) For determination of surface coverage, cf.: Katz, E.; Solov'ev, A. A. J. Electroanal. Chem. 1990,291, 171. (12) A closely related monolayer of dinitro spiropyran was prepared by covalent linkage of 3 to a monolayer generated by reaction of (3-aminopropy1)triethoxysilane to an I T 0 transparent electrode. The resulting spiropyran monolayer exhibits the characteristic reversible photoisomerizable properties that were followed spectroscopically. The electrochemical properties of the I T 0 electrode toward the DNP-Ab are similar to those described for the Au electrode but are lessaccuratedue to their variablesurfaceareas. Accordingly, we preferred to report the resultson Au electrodes that exhibit constant surface areas.
0002-7863/94/1516-9365$04.50/00 1994 American Chemical Society
Communications to the Editor
9366 J. Am. Chem. SOC.,Vol. 116, No. 20, 1994 Scheme 1. Organization of a Photoisomerizable Dinitro Spiropyran Antigen Monolayer Electrode
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of DNP-Ab is shown in Figure 3. The 4a monolayer electrode is insulated by the antibody and reveals affinity toward DNPAb. Thdbmonolayer electrodedoesnot interact with the DNPAb, and the amperometricresponse of the electrode is not altered in the presence of the Ab. Thus, the dinitrophenylunit included in the 4a spiropyran monolayer electrode acts as antigen for the DNP-Ab, while the 4b merocyanine monolayer lacks antigen properties. Figure 3 (inset) shows the reversible activity of the dinitro spiropyran monolayer electrode as amperometric sensor for the DNP-Ab. The experiment is initiated with the 4b monolayer electrode. The amperometricresponse in the presence of the DNP-Ab is similar to that of the electrode in the absence of the DNP-Ab. The high amperometric response implies that the Ab does not associate with the monolayer electrode. 11lumination of the 4b monolayer electrode, X > 495 nm, results in the 4th monolayer electrode.13 Incubation of the 4a monolayer electrodewith the DNP-Ab (8 nM, 15 min) results in a decrease in the amperometric signal of the electrode, implying association of the DNP-Ab with the electrode. Further irradiation, X = 360-380 nm, of the 4a monolayer electrode that was interacted with the DNP-Ab results in the 4b monolayer electrode. The (1 3) The amperometric response of the photogenerated 4a monolayer electrode in the absence of the DNP-Ab is identical to that of the 4b monolayer electrode. (14) It should be noted that only ca. 50% of the original amperometric response is restored, implying only partial dissociation of the DNP-Ab. This is attributed to incomplete isomerizationof the 4n monolayer to 4b monolayer electrode. The electrode revealed eight reversible cycles of similar amperometricresponsesobservedinthe first cycle (5096oftheinitialvalue). Prolonged irradiation of the 4. monolayer electrode increased the conversion yield to 4b monolayer, and higher amperometricresponseswere detected due to enhanced release of the DNP-Ab. Prolonged irradiation of the electrode, however, acts adversely on the cyclic activity of the electrode, presumably due to partial decomposition of the dinitro spiropyran monolayer.
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E (V) Figure 3. Cyclic voltammograms of the 4a monolayer electrode in the presence of anti-DNP antibody, IO pM,at different times of electrode treatment: (I) after 0 min, (2) after 3 min, (3) after 11 min, and (4) after 16 min. The 4a electrode was produced by continuous irradiation at X > 495 nm. (Inset) Cyclic amperometric responses of the 4 " i f i e d monolayer electrode in the presence of anti-DNP antibody, 1.5 p M (0) 4b monolayer electrode; (0) 41 monolayer electrode. The configuration of the 4b monolayer electrode was generated by illumination of the 4a monolayer electrode at 360 < X 380 nm. The configuration of the 4a monolayer electrode was produced by illumination of the 4b monolayer electrode at X > 495 nm. All experiments were recorded in an electrochemical cell that consisted of PBS buffer and 1.1 mM KJ[Fe(CN)6] (37 f 2 "c), the antigen monolayer electrode (3 X lo-* cm2) as working electrode, Pt wire as counter electrode, and Ag/AgCl as reference electrode.
rinsed electrodeyields a high amperometric response, indicating that the DNP-Ab that was originally associated with theantigenic monolayer electrodewas washed off. This amperometricresponse is not altered upon addition of the DNP-Ab. Subsequent photochemical reisomerization of the 4b monolayer to the 4a monolayer electrode results in a low amperometric signal upon interaction of the resulting electrode with the DNP-Ab. Thus, the activity of the monolayer electrode toward sensing the DNPAb is regenerated, and the two-step illumination of the photoisomerizable antigen monolayer electrode enables its reversible cyclic performance.14 We conclude that antigen monolayer electrodes provide a sensitive interface for amperometric detection of the complementary antibody.15 Photoisomerizable antigenic monolayers highlight a general means for cyclic operation of amperometric immunosensors. Acknowledgment. This research was supported by a grant from the Ministry of Science and Technology, Israel, and the Commission of the European Communities. (15) Israel Patent Application No. 108726, filed on Feb 22, 1994.
